Mechanically reinforced biotubes for arterial replacement and arteriovenous grafting inspired by architectural engineering

There is a lack in clinically-suitable vascular grafts. Biotubes, prepared using in vivo tissue engineering, show potential for vascular regeneration. However, their mechanical strength is typically poor. Inspired by architectural design of steel fiber reinforcement of concrete for tunnel construction, poly(ε-caprolactone) (PCL) fiber skeletons (PSs) were fabricated by melt-spinning and heat treatment. The PSs were subcutaneously embedded to induce the assembly of host cells and extracellular matrix to obtain PS-reinforced biotubes (PBs). Heat-treated medium-fiber-angle PB (hMPB) demonstrated superior performance when evaluated by in vitro mechanical testing and following implantation in rat abdominal artery replacement models. hMPBs were further evaluated in canine peripheral arterial replacement and sheep arteriovenous graft models. Overall, hMPB demonstrated appropriate mechanics, puncture resistance, rapid hemostasis, vascular regeneration, and long-term patency, without incidence of luminal expansion or intimal hyperplasia. These optimized hMPB properties show promise as an alternatives to autologous vessels in clinical applications.

rings which were clamped by machine chucks, and then pulled radially at a rate of 10 mm/min until rupture. Tensile strength and ultimate elongation at break were measured. The tensile strength was set as the peak stress of the stress-strain curve. Young's modulus was calculated from the initial linear region (up to 5% strain) of the stress-strain curve.
For samples obtained from sheep, strength after repeated puncture and suture retention strength was tested in accordance with ISO 7198:2016. Briefly, heat-treated medium-fiberangle PB (hMPB) was punctured 0, 8, 16 and 24 times with a 16G dialysis needle in accordance with ISO 7198:2016 A.5.2.7 "strength after repeated puncture". Then, radial tensile strength was tested as above. Suture retention strength, including straight-across and oblique procedures, was conducted in accordance with ISO7198:2016 A.5.7 "suture retention strength". The straight-across procedure to assess suture retention followed the same method used to assess the samples obtained from rats. For the oblique procedure, hMPB was cut at an angle of 45° to the axis, and at the point 2 mm from the cut surface, a 6-0 prolene suture was pushed through the hMPB piece. The hMPB and the suture wire were fixed on a tensile testing machine and pulled with the same parameters as the straight-across procedure at three points of the hMPB piecetoe, 90°, and heel positions. Mechanical tests were performed in quintuplicate.

Vascular function
After hMPBs were implanted into rat abdominal arteries for 3 months and canine carotid arteries for 7 months, the physiological function of regenerated and native arteries was assessed by aortic ring bioassay using a PowerLab/870 Eight-channel 100kHz A/D converter (AD Instruments, Sydney, Australia). Sample rings of 3 mm length were submerged in Krebs buffer (composition in mM: NaCl,118.4;KCl,4.7; CaCl 2 , 2.5; MgSO 4 , 1.2; KH 2 PO 4 , 1.2; NaHCO 3 , 25; dextrose, 11.1; Na 2 Ca EDTA, 0.029; pH 7.4) at 37 ℃ and gassed with carbogenic mixture (95% O 2 and 5% CO 2 ). All preparations were stabilized under a resting tension of 2 g for 1 h with the buffer changed every 15 min. The presence of functional smooth muscle cells was indicated by the contractile responses induced by addition of potassium chloride (KCl) (60 mM). The function of the neo-endothelium was confirmed by the relaxation induced by acetylcholine (ACh) (10 μM), in pre-constricted segments by adrenaline (AD) (1 μM), and vascular smooth muscle function was evaluated by vascular relaxation in response to sodium nitroprusside (SNP) (1 × 10 -7 mol/L). Isometric forces were recorded with force transducers connected to a PowerLab/870 Eight-channel 100 kHz A/D converter (AD Instruments). The physiological function of as prepared hMPBs in subcutis of rats and canine models, and expanded polytetrafluoroethylene (ePTFE) grafts after implantation into canine carotid artery for 7 months was measured using the same protocol.

Western blot
Western blot was performed to semi-quantify α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (MYH) protein expression of hMPBs before and after implantation into canine carotid artery for 7 months. In brief, total protein was extracted from samples using a Tissue Protein Extraction kit (Thermo Fisher Scientific, USA) containing protease inhibitors. The lysates were centrifuged at 12,000 rpm for 10 min at 4°C and the supernatants were transferred to fresh 1.5 mL tubes. Protein concentrations were determined by BCA assay (Solarbio, China). After boiling for 5 min with SDS-PAGE loading buffer, the protein samples were separated by electrophoresis using 4-12% SDS-PAGE gels. The separated proteins were transferred to a PVDF membrane (Merck Millipore, USA) and incubated with the appropriate primary antibodies overnight at 4°C . Primary antibodies were mouse anti-α-SMA (Abcam, ab7817, 1:1000), rabbit anti-MYH (Abcam, ab212657, 1:2000), and mouse anti-β-actin (Abcam, ab6276, 1:5000). The membranes were washed five times with PBS-Tween (PBST) before incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (HRP-goat anti-mouse IgG (H+L) antibody, Bioworld, bs12478, 1:5000; or HRP-goat anti-rabbit IgG (H+L) antibody, Bioworld, bs13278, 1:5000) for 2 h at room temperature. Following a further six washes with PBST, bound antibodies were detected using the Immobilon Western HRP detection reagents (Merck Millipore) and a Tanon-5500 Chemiluminescent Imaging System (Tanon, China). The intensities of all bands were quantified using Image-Pro Plus 6.0 software. ePTFE grafts implanted into canine carotid artery (cCA) for 7 months and native cCA tissue were used as controls and assessed using the same protocol.

Quantitative analysis of neointima thickness
Neointima thickness was measured using Image-Pro Plus 6.0 software based on hematoxylin and eosin (H&E) stained sections. For rats, three high-magnification images per sample and five samples per group were included to obtain the statistical results. For canine, three whole cross-sections images per sample and three samples per group were included to obtain the statistical results. For sheep, four low-magnification images per section, three sections per sample and three samples per group were included to obtain the statistical results.

Quantitative analysis of endothelial cells (ECs) coverage rate
For rats, the ECs coverage rate was quantified by adding the length of endothelial nitric oxide synthase (eNOS)-positive monolayer and dividing this sum by the length of the longitudinal section of the hMPBs. Three sections per sample and five samples per group were included to obtain the statistical results.

Quantitative analysis of contractile SMCs thickness
For rats, the contractile smooth muscle cells (SMCs) thickness was analysed by immunofluorescence (IF) staining of the cross sections of the explanted hMPBs using anti-MYH antibody. Four high-magnification images per section, three sections per sample and five samples per group were included to obtain the statistical results. For canine, the contractile SMCs thickness was analysed by immunohistochemistry (IHC) staining of the cross sections of the explanted hMPBs using anti-MYH antibody. Four high-magnification images per sections, three sections per sample and three samples per group were included to obtain the statistical results. For sheep, the contractile SMCs thickness was analysed by IF staining of the cross sections of the explanted hMPBs using calponin (CNN) antibody. Four low-magnification images per sections, three sections per sample and three samples per group were included to obtain statistical results.

Quantitative analysis of CD68 + macrophage number
After cross-sections of samples were stained with anti-CD68 antibody, all CD68 + cells within low-magnification images were counted to assess the inflammatory state of tissue samples.
For rat tissue sections, four images per section, three sections per sample and five samples per group were included to obtain the statistical results. For canine and sheep, four images per section, three sections per sample and three samples per group were included to obtain the statistical results.

Quantitative analysis of micro-vessels formation
After cross-sections of samples were stained with anti--SMA, all -SMA + microvasculature within low-magnification images were counted to assess micro-vessels formation. For rat tissue sections, four images per section, three sections per sample and five samples per group were included to obtain the statistical results. For canine and sheep, four images per section, three sections per sample and three samples per group were included to obtain the statistical results.

Detection of bacterial infection in hMPB arteriovenous grafts (AVG) in sheep models
The corresponding tissue samples were weighed and homogenized using a tissue homogenizer (Heidolph Diax 900) in 10 mL buffered peptone water/g sample. A total volume of 100 μL of homogenate was plated and spread onto standard agar plates. Colony growth was observed after incubation at 37℃ overnight. In addition, 100 μL of the homogenate was added to Luria-Bertani (LB) liquid medium, incubated at 37℃ with an orbital shaker speed of